The cyclin-dependent kinase inhibitor p27 is required for an effective cell cycle arrest in vivo. This arrest is relieved by degradation of p27 via the ubiquitin-dependent proteolysis system. Depletion of the p27 tumor suppressor resulting form hyperproteolysis is a hallmark of advanced carcinomas that correlates with decreased survival. P27 degradation is mediated by SCF:SKP2-dependent polyubiquitylation and SKP2 overexpression is sufficient to cause ectopic p27 degradation. Hyperactivation of the SKP2-mediated proteolysis pathway, therefore, appears to be the main mechanism of p27 downregulation in carcinomas and a good target for therapeutic intervention. We are proposing a chemical biology approach to identify synthetic molecules able to interfere with SKP2-dependent p27 proteolysis. The biochemical understanding of this pathway is now at a point where screens for specific inhibitors can be designed. Small molecules able to interfere with SKP2-dependent p27 degradation are predicted to restore p27 expression in carcinomas thus inhibiting their unrestrained growth. This prediction constitutes the principal hypothesis of this grant application. Specifically targeting this distinct defect for therapy was never before attempted. Considering that virtually all normal differentiated cells express high levels of p27, such compounds bear a high potential for tumor specificity, thus minimizing drastic adverse effects. The goal of this project is to identify "SMIPs", small molecules that interfere with SKP2-mediated p27 proteolysis. To this end, we will use the following approaches: A.1. Optimize an in vitro assay to screen synthetic chemical libraries for inhibitors of the SKP2/p27 protein interaction. A.2. Develop a cell-based assay to screen for inhibitors of SKP2-mediated p27 degradation in vivo. A.3. Test putative SMIPs identified in the primary screens in additional in vivo and in vitro assays, which address both their specificity and efficacy for inhibition of p27 degradation.